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Cholesterol and phospholipids in cultured skin fibroblasts from patients with dystonia.

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BRIEF COMMUNICATIONS AND CASE REPORTS
Cholesterol and
Phospholipids in Cultured
Skm Fibroblasts from
Patients with Dystonia
William A. Maltese, PhD, and Darryl C. De Vivo, M D
Cultured skin fibroblasts from patients with hereditary
(recessive) torsion dystonia or adult-onset sporadic dystonia and from healthy volunteers were compared with
respect to total cellular cholesterol, total phospholipid,
and phospholipid composition. Differences that could be
attributed specifically to dystonia were not detected.
The data suggest that the reported cell membrane abnormality in recessive torsion dystonia does not involve a
generalized defect in cholesterol or phospholipid metabolism.
Maltese WA, De Vivo DC: Cholesterol
and phospholipids in cultured skin
fibroblasts from patients with dystonia.
Ann Neurol 16:250-252, 1984
The biochemical abnormalities underlying the idiopathic and hereditary forms of torsion dystonia are obscure. Preliminary data have suggested the existence of
a generalized cell membrane abnormality in the recessive form of dystonia [S}, but the nature of this abnormality has not been defined. The molar ratio of cholesterol to phospholipid and the relative proportions of
the different phospholipid classes are major determinants of membrane fluidity and lipid-protein interaction. Changes in lipid composition can affect several
membrane processes that are crucial to neural function,
such as ionic flux [7], orientation and/or affinity of
serotonin receptors [4, 71,acetylcholinesterase activity
[3], and formation of neurites I S ] . For these reasons,
the possibility of a lipid defect in dystonia merits investigation.
Methods
Skin fibroblast cultures were derived from healthy volunteers
and patients evaluated at the Dystonia Clinical Research Center at the Columbia-Presbyterian Medical Center. Informed
From the Pediatric Neurology Divisional Laboratories and the Dystonia Clinical Research Center, Department of Neurology, College
of Physicians and Surgeons of Columbia University and the New
York State Psychiatric Institute, New York, N Y 10032.
Received Oct 26, 1983, and in revised form Jan 10, 1984. Accepted
for publication Jan IS, 1984.
Address reprint requests to Dr Maltese, Department of Neurology,
College of Physicians and Surgeons of Columbia University, 710 W
168th St, New York, N Y 10032.
250
consent was obtained from all participants in the study. Cells
were maintained at 37°C in Dulbecco's modified Eagle medium, supplemented with 10% (v/v) fetal calf serum, and
equilibrated with 5% carbon dioxide in air. Cultures used for
lipid analyses were between the ninth and eleventh passages
and were confluent at the time of harvest. Similar saturation
densities (expressed as total protein per culture) were attained by cells from controls and patients with dystonia. Delipidated serum was prepared by extraction with butanolldiisopropyl ether (2:3, v/v) as described elsewhere 121. Protein
determinations were performed by means of a microbiuret
method 161, using bovine serum albumin as the standard.
For lipid analyses cells from two 150 cm2 cultures were
pooled and suspended in 0.15 M sodium chloride. Lipids
were extracted with methanol and chloroform (saline/
methanollchloroform, 3 :4 :8), and aliquots of the lower
phase were dried under nitrogen. Cholesterol was assayed by
a colorimetric method, using a commercial kit (Stanbio Laboratory, San Antonio, TX), and lipid phosphorus was measured according to the method of Anderson and Davis C11.
Individual phospholipids were separated by two-dimensional
thin layer chromatography on silica gel G plates, using solvent systems consisting of chloroforndpetroleum ether/
methanoVglacia1 acetic acid (5: 3: 1.6:1 ) and chloroform'
acetone/methanoUglacial acetic acid/water (5 : 2 : 1: 1:0.5).
Phospholipid spots, visualized by exposing the plates to
iodine vapor, were identified on the basis of their mobilities
relative to phospholipid standards (Supelco, Bellefonte, PA).
Lipid zones were eluted with chloroforndmethanol(2: I), and
phosphorus values for individual phospholipids were expressed as percentages of the total phosphorus recovered in
all spots.
Results
Fibroblast cultures from a total of ten patients with
dystonia, five healthy volunteers, and two unaffected
parents of patients with dystonia had similar concentrations of total cholesterol and phospholipid in fibroblast
cultures (Table). Because lipids present in fetal calf
serum can contribute to the cellular lipid profile, a defect in one of the lipid biosynthetic pathways might not
result in an altered fibroblast lipid composition unless
all exogenous lipids were removed from the culture
medium. To test this possibility, fibroblasts were grown
for one generation in medium supplemented with delipidated fetal calf serum prior to being harvested for
lipid analyses. As in the case of cells grown with whale
serum, similar cholesterol and phospholipid concentrations were measured in cultures from patients with dystonia and controls (see the Table). Further analysis of
phospholipids separated by two-dimensional thin layer
chromatography failed to reveal any consistent deficiencies that could be attributed specifically to the
derivation of cell lines from patients with dystonia (see
the Table).
Discussion
The data suggest that radical generalized defects in the
cellular pathways for synthesis andor uptake of choles-
Total Cholesterol, Total Phospholipid, and Phospholipid Composition of Cultured Skin Fibroblasts
Patients with
Adult-Onset
Dystoniab
Patients with
Childhood-Onset
Dystonia'
Unaffected
Parents
*
5
112.2 -+ 11.1
213.5 t 6.3
0.525
0.049
*
5
127.0
12.2
219.7 t 16.3
0.577 t 0.033
2
133.1 k 10.5
226.1 t 22.3
0.590 t 0.012
5
147.4 IC_ 13.1
252.8 f 5.0
0.585 t 0.056
5
127.3 t 3.2
244.5 t 8.9
0.524 t 0.028
5
151.6 f 13.7
241.4 +- 3.4
0.627 -t 0.055
2
160.9 t 13.3
234.9 t 10.9
0.689 t 0.089
5
3
2
49.3 t 1.3
24.4 t 1.2
5.6 -t- 0.1
9.8 2 0.2
10.9 f 0.9
45.8
25.3
8.1
9.4
11.4
3
5
3
45.6 t 2.8
29.5 2 0.8
5.4 2 0.5
8.8 t 0.4
10.6 2 2.6
50.2 Tt_ 1.1
24.2 t 2.4
8.3 k 0.2
8.1 2 0.7
9.1 t 0.7
3.4
$3.5
25.4
5.0
4.6 t 0.2
8.1 t 0.7
8.4 -+ 1.2
Healthy
Volunteersa
Variable
CELLS GROWN WITH WHOLE FETAL CALF SERUM
No. of subjects
Total cholesterold
Total phospholipide
U P molar ratio
5
129.6 t 3.7
222.3 f 6.1
0.585
0.025
*
CELLS GROWN WITH DELIPIDATED SERUM
No. of subjects
Total cholesterold
Total phospholipid'
U P molar ratio
CELLS GROWN WITH WHOLE FETAL CALF SERUM
No. of subjects
Phospholipid compositionf
Phosphatid ylcholine
Phosphatid ylethanolamine
Phosphatid ylserine
Phosphatid ylinositol
Sphingomyelin
3
47.9
28.2
4.6
9.5
9.8
-t
?
?
-t
2
1.4
0.1
0.3
0.4
1.3
t
t
t
t
t
1.0
1.6
0.8
0.5
2.1
42.6 t 2.4
30.1
0.6
5.5 2 1.3
10.3 t 1.2
11.6 +- 0.5
*
CELLS GROWN WITH DELIPIDATED SERUM
No. of subjects
Phospholipid composition'
Phosphatidy lcholine
Phosphatid ylethanolamine
Phosphatidylserine
Phosphatidylinositol
Sphingomyelin
~
~~
~
~~~
~
~~~
2
*
*
~
45.2
30.4
6.1
10.3
8.2
t 1.2
+- 3.5
t 0.4
k
0.5
t 1.5
~~
"Average age r+_ SD, 37
4 years (n = 5 ) .
bIncludes four patients with sporadic dystonia and one patient with tardive dystonia; average age 2 SEM, 45 ? 5 years.
'All patients had positive family histories of dystonia following an autosomd recessive pattern; average age Ifr SD, 38 Ifr 4 years (n = 5 )
dExpressed as nmoVmg cell protein (mean ? SEM).
'Expressed as nmol lipid phosphorudmg cell protein (mean % SEM).
'Expressed as percentage of total lipid phosphorus eluted in all phospholipid spots from the chromatogram (mean -t SEM).
UP = CholesteroYphospholipid.
terol and phospholipids do not contribute to the
biochemical abnormalities of the sporadic and recessive
forms of torsion dystonia. Whether this probability also
applies to dystonias that appear to follow a dominant
pattern of inheritance remains to be determined. The
data do not preclude the possibility of alterations in the
fatty acid compositions of individual phospholipids, nor
can studies of fibroblasts rule out the possibility that
dystonia involves a defect in the metabolism of lipids,
such as galactocerebrosides and sulfatides, that are synthesized primarily in the nervous system. Although our
studies were performed with whole cells, the lipid
profile of cells grown in lipid-depleted medium should
reflect the lipid composition of the cell membrane network. Thus, the data imply that the membrane abnormalities previously observed in cells from patients with
dystonia [S] are not due to major alterations in the
cholesteroVphospholipid molar ratio or in phospholipid composition.
Supported by grants from the Dystonia Medical Research Foundation and the Kathryn and Gilbert Miller Fund for Child Neurology
Research.
The authors thank Diane Doucette and Christina Pansarasa for excellent technical assistance, Carol Moskowitz for collection of skin
biopsy specimens, and Dr Stanley Fahn for helpful comments.
References
1. Anderson RL, Davis S: An organic phosphorus assay which
avoids the use of hazardous perchloric acid. Clin Chim Acta
121:111-116, 1982
Brief Communication: Maltese and De Vivo: Lipids in Dystonia Fibroblasts 251
2. Cham BE, Knowles BR: A solvent system for delipidation of
plasma or serum without protein precipitation. J Lipid Res
17:1 76- I 8 1, 1976
3. Farias R, Bloj B, Morero RD, et al: Regulation of allosteric memhrane-bound enzymes through changes in membrane lipid composition. Biochim Biophys Acta 415:231-252, 1975
4. Heron DS, Shinitzky M, Hershkowitz M, Samuel D: Lipid fluidity markedly modulates the binding of serotonin to mouse membranes. Proc Natl Acad Sci USA 77:7463-7467, 1980
5 . Maltese WA, Reirz BA, Volpe JJ: Effects of prior sterol depletion
on neurite outgrowth in neurohlastoma cells. J Cell Physiol
108:475-482, 1981
6. Munkres KO, Richards FM: The purification and properties of
NeuroJporu malate dehydrogenase. Arch Biochem Biophys
1O9:466-4 79, 1965
7. Papaphilis A, Deliconstanrinos G: Modulation of serotonergic
receptors by exogenous cholesterol in dog synaptosomal membrane. Biochem Pharmacol 29:3325-3327, 1980
8. Pettegrew JW, Minshew NJ, Stewart RM: Furrher evidence of a
membrane abnormality in inherited dystonia (abstract). Ann
Neurol 10:78, 1981
9 . Wiley JS, Cooper RA: Inhibition of cation cotransport by cholesterol enrichment of human red cell membranes. Biochim Biophys
Acta 413:425-431, 1975
Nonconvulsive Status
Epilepticus Following
Metrizamide Myelography
Paul B. Pritchard 111, MD, and David B. ONeal, M D
Epileptic seizures are a known complication of metrizamide myelography. To our knowledge, this is the
first report of a case of nonconvulsive status epilepticus of the absence type following metrizamide
myelography. There was symptomatic and electroencephalographic improvement after intravenous administration of antiepileptic drugs, and there was no
neurological residual. Nonconvulsive status epilepticus
should b e considered when impairment of consciousness
supervenes after radiographic procedures using metrizamide.
Pritchard PB 111, O N e a l DB: Nonconvulsive status
epilepticus following metrizamide myelography.
Ann Neurol 16:252-254, 1984
Several neurological complications of metrizamide myelography have been reported, including epileptic sei-
-
From the Neurology Service, VA Medical Center, and the Department of Neurology, Medical University of South Carolina, Charleston, SC.
Received Jan 9, 1984. Accepted for publication Jan 14, 1984.
Address reprint requests to Dr Pritchard, Neurology Service, VA
Medical Center, 109 Bee Street, Charleston, SC 29403.
252
zures 19, 101, asterixis and diffuse encephalopathy [l],
meningeal irritation 14}, a n d areflexia [21. To o u r
knowledge, this is the first reported case of nonconvulsive status epilepticus of t h e absence type following
metrizamide myelography.
Case Report
A 59-year-old man was admitted to VA Medical Center,
Charleston, for metrizamide myelography because of slowly
progressive spastic paraparesis. An earlier computed tomographic (CT) scan and an iophendylate myelogram had failed
to demonstrate a definitive abnormality.
Ten ml of metrizamide (Amipaque, 300 mg iodine per
milliliter) was introduced into the lumbar subarachnoid space
at the L3 level and taken to the cervicocranial junction. Myelography demonstrated a small posterior osteophyte at the
C4-5 level, and a C T scan showed no additional abnormality.
Three hours later the patient became nauseated, vomited,
and appeared confused. He attended to visual stimuli, but his
only response to questions was “huh” or “uh-huh.” The outstretched arms showed a fine tremor of the fingers and slight
asterixis of the hands. There were no changes in the previously noted motor signs, nor was there any additional focal
neurological deficit. Serum electrolytes and metabolic studies
were normal except for minimal hypokalemia.
Video electroencephalogram (EEG) performed the following day, at which time the patient was still severely confused.
showed continuous 2-Hz sharp-slow wave discharges up to
300 p V in amplitude with generalized distribution but ante-rior predominance (Fig I), superimposed on moderate diffuse slowing of background activity. Apart from regional predominance of discharges, no focal abnormality was evident.
The discharges were continuous and were not affected by eye
opening or closure.
Three intravenous injections of diazepam, totaling 8 mg,
were given. Shortly thereafter, the patient responded to
questions more appropriately, with brief phrases. He knew
that he was in the hospital, and he recognized his physicians.
EEG (Fig 2) demonstrated attenuation of discharges and induction of beta activity. A few minutes later, continuous
sharp-slow wave discharges reappeared bilaterally, and the
patient was given 1,100 mg of phenytoin intravenously. The
EEG resumed the appearance shown in Figure 2 shortly
thereafter.
Two days later, the patient had returned to his neurological
baseline. He was amnesic for most events of the previous 4 8
hours. He was not given maintenance antiepileptic drugs, and
he had no further seizures over the subsequent eleven
months. An EEG two months later was unremarkable except
for rare temporal sharp waves of dubious clinical importance.
Discussion
The clinical and EEG differentiation between “absence
status” and ‘‘temporal lobe status” has been delineated
15-71. B y t h e established criteria based on characteristic EEG and clinical features, the findings in our patient
are consistent with absence status. Absence status in
adults, also known as spike and wave stupor [S] and
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